The present invention is directed to modified proteins and methods of producing the same. More specifically, the modified protein of the present invention is a target protein into which is inserted a controllable intervening protein sequence (CIVPS), the CIVPS being capable of excision under predetermined conditions.
Production of mature proteins involves the flow of information from DNA to RNA to protein. Precise excision of DNA and RNA elements which interrupt that information has been previously described [M. Belfort, Annu. Rev. Genet. 24, 363 (1990); T. R. Cech, Annu. Rev. Biochem. 59, 543 (1990); Hunter et al., Genes Dev. 3, 2101 (1989)]. More recently, evidence for the precise excision of intervening protein sequences has also been described for the TFPI allele from Saccharomyces cerevisiae [Hirata et al., J. Biol. Chem. 265, 6726 (1990); Kane et al., Science 250, 651 (1990)] and the rec A gene from Mycobacterium tuberculosis [Davis et al., J. Bact. 173, 5653 (1991); Davis et al., Cell 71:1 (1992)]. Each contains internal in-frame peptide segments which must be removed to produce the mature protein. Expression of Tfp1 and Rec A each results in two peptides: one representing the intervening protein sequence (IVPS) and the other the ligated product of the external protein sequences (EPS). This post-translational processing event has been termed "protein splicing". Similarly, the Vent DNA polymerase gene from the hyperthermophilic archaea Thermococcus litoralis contains two in-frame IVPSs [Perler, et al., PNAS 89, 5577 (1992)].
A major impediment to the development of methods of using IVPSs or protein splicing in other than research applications has been the inability to control the activity of the IVPS and thus the splicing event.
Thus, it would be desirable to have a method which provides a ready means to modify a target protein using an IVPS, particularly where the activity of the IVPS is controllable. It would also be desirable to have a method which can specifically modify target proteins such that their activity is substantially inactivated. It would be desirable to have a method which can be used to restore the activity of an inactivated modified protein.